An effect of fluorescent probes and of insulin on the structure of adipocyte membranes [proceedings

Abstract

Fluorescent probes can be used to monitor changes in the structure of cell membranes (Radda & Vanderlooi, 1972). In the present study 8-anilinonaphthalene-lsulphonic acid, 12-(9-anthroyI)stearate and N-phenylnaphthylamine have been mixed with fragments of plasma membrane prepared from rat adipocytes (Rodbell, 1967 ; Czech & Lynn, 1973). The intensity and polarization of the resulting fluorescence have been recorded. Fluorescence measurements were made with a Hitachi MPF 2A spectrofluorimeter or with a laboratory-built split-beam fluorimeter of high sensitivity. The addition of insulin (5-50011~) to the probe-labelled membrane suspension caused no change in the fluorescence characteristics of the probes. However, preincubation of a membrane suspension with insulin increased the rate of change of the polarization of anthroyl stearate fluorescence. This rate was measured by recording continuously the fluorescence polarization observed after the addition of anthroylstearate to the suspension. The fluorescence polarization observed after preincubation of the membranes with insulin at concentrations of 50n~ and l5On~ is shown in Fig. 1 together with the polarization observed with control membranes. This shows that insulin affects the gross structure of the membrane. We suggest that the reason for failing to detect an effect of insulin on membranes already interacted with probe is that the probes themselves cause a change in the membrane structure. Evidence for this hypothesis is of two kinds. The fluorescence polarization of anthroylstearate interaction with membranes is concentration-dependent . Anthroylstearate was added to different batches of a membrane suspension to give final concentrations of 200,~~ and 400~~. The fluorescence